MinElute Reaction Cleanup Kit (250)

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Publication protocol

Amplicon preparation and sequencing on GS Junior (Roche).: The PCR was performed in a total volume of 20 µl containing 50 ng of template DNA, 0.5µM of each primer (without barcode), 200µM each dNTP, 2mM MgCl2, 1 x Phusion HF Buffer, and 0.4 U of Phusion HF DNA Polymerase (Thermo Scientific). Amplification was carried out using 2 step protocol with initial denaturation at 98°C for 30s, followed by 35 cycles of 98°C for 10s and 72°C for 10s, and final extension at 72°C for 5 min. Negative control containing miliq H2O instead of template DNA was included in parallel. PCR products were visualized on 1 % agarose gel and cleaned using MinElute Reaction Cleanup Kit (QIAGEN). A second PCR was performed under the same conditions by using barcoded primers and 1 µl of cleaned amplicon as a template. Each sample was done in triplicate, PCR products were pooled, run on 1% agarose gel and 450bp fragment was cut and purified from the gel using GFX PCR DNA and Gel Band purification kit (GE Healthcare). Samples were eluted in 40µl of Tris-HCl and amplicon concentration was measured using Qubit dsDNA HS Assay Kit (life technologies). Two amplicon pools were created with 5 samples each by pooling them in equimolar concentrations. Five hundred ng of each pool was purified with MinElute Reaction Cleanup Kit (QIAGEN) and eluted in 16ul TE buffer. Amplicon sequencing was performed using GS Junior (Roche) at former MTT Agrifood Research Finland following the standard procedures.


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